Regulated proteolysis by the ubiquitin-proteasome system (ubiquitin system) plays essential roles in a multitude of biological processes and has major ramifications for human health and disease, including illnesses that range from cancer and neurodegeneration to cardiovascular syndromes and defects of immunity. Our studies of the ubiquitin system and the ubiquitin-dependent N-end rule pathway over the last three decades were made possible, to a large extent, by the present grant (GM031530), currently in its 33rd year of support. The N-end rule pathway recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins and thereby causes their degradation by the proteasome. Recognition components of the N-end rule pathway are called N-recognins. In eukaryotes, N-recognins are E3 ubiquitin ligases that can target N-degrons. The N-end rule pathway consists of two major branches. The first branch, called the Arg/N-end rule pathway, targets specific unacetylated N-terminal residues of protein substrates. This branch, discovered by our laboratory in 1986, continues to be a fount of biological insights and remains a focus of our studies. The other branch, called the Ac/N-end rule pathway, was discovered by our laboratory in 2010. This pathway recognizes proteins that bear N-terminally acetylated residues. Our recent (2014) study has revealed a specific complementarity (functional coupling) between the Arg/N-end rule and Ac/N-end rule pathways. The present (GM031530) renewal application focuses on this physiologically important coupling as well as the Ac/N-end rule pathway itself. The proposed projects include both functional and mechanistic studies of specific physiological substrates of the mammalian (mouse) Ac/N-end rule pathway, including Rgs2 (a regulator of signal transduction) and Aanat (serotonin N-acetyltransferase, a key circadian enzyme). These studies also include a detailed dissection of the targeting specificity of both previously identified and recently discovered N-recognins of the N-end rule pathway, including Not4, Doa10, and Ubr1 in the yeast Saccharomyces cerevisiae as well as the larger set of mammalian (mouse) counterparts of yeast N-recognins. The proposed studies will advance the understanding of the N-end rule pathway, a multifunctional, highly regulated, universally present and medically significant biological system.